A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment Induces Pericyte and IgA-Dependent Wound Repair and Lifespan Extension in a Colon Tumor Model.

Environmental enrichment (EE) replicates mind-body therapy by providing complex housing to laboratory animals to improve their activity levels, behavior, and social interactions. Using a Tcf4Het/+ApcMin/+-mediated model of colon tumorigenesis, we found that EE vastly improved the survival of tumor-bearing animals, with differential effect on tumor load in male compared to female animals. Analysis of Tcf4Het/+ApcMin/+ males showed drastically reduced expression of circulating inflammatory cytokines and induced nuclear hormone receptor (NHR) signaling, both of which are common in the wound repair process. Interestingly, EE provoked tumor wound repair resolution through revascularization, plasma cell recruitment and IgA secretion, replacement of glandular tumor structures with pericytes in a process reminiscent of scarring, and normalization of microbiota. These EE-dependent changes likely underlie the profound improvement in survival of colon-tumor-bearing Tcf4Het/+ApcMin/+ males. Our studies highlight the exciting promise of EE in the design of future therapeutic strategies for colon cancer patients.

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Harnessing genetic diversity in Saccharomyces cerevisiae for fermentation of xylose in hydrolysates of alkaline hydrogen peroxide-pretreated biomass.

The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na(+), acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX™ pretreated corn stover.

BACKGROUND: Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products. RESULTS: In this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEX™ (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35°C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain. CONCLUSION: These results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.